C Neoformans Essay, Research Paper
BackgroundThe organism C neoformans is an encapsulated yeast; its environmental niche has not been completely defined, although outbreaks of disease have been associated in particular with pigeon roosts and other large contaminated sites. There are two varieties of C neoformans, distinguished by antigenic differences in the outer capsule of the organism: serotypes A and D (C neoformans var neoformans, the most common strain) and serotypes B and C (C neoformans var gatti). Cryptococcus neoformans var neoformans is the principal pathogen in patients with AIDS. Cryptococcus neoformans var gatti, which is found predominantly in Australia, Asia, and Southern California, has only rarely been implicated to cause disease in this population.
Antigenic specificity of the capsular polysaccharides define the four different serotypes of C. neoformans; A, B, C and D. C. neoformans variety neoformans includes serotypes A and D while C. neoformans var. gattii are associated with serotypes B and C. Worldwide, most Cryptococcal infections in immunocompromised patients, including HIV infected patients, are due to C. neoformans var. neoformans, serotype A. In a smaller number of immunocompromised patients, infecton is caused by C. neoformans var. neoformans, Serotype D. For unknown reasons, HIV infected patients and other immunosuppressed patients are rarely infected with the variety gattii. Patients infected with C. neoformans var. gattii are usually immunocompetent, respond slowly to treatment, and are at risk for developing intracerebral mass lesions (cryptococcomas).
Cryptococcus neoformans reproduces by budding and forms round yeast-like cells 3-6 micrometers in diameter. Within the host and in certain culture media, a large polysaccharide capsule surrounds each cell. Cryptococcus neoformans forms smooth, convex, yellow or tan colonies on solid media at 20 to 37 degrees Celsius. The identification of this fungus is based on its microscopic appearance, biochemical tests, and its ability to grow at 37 degrees Celsius. Most nonpathogenic strains of Cryptococcus do not grow at 37 degree Celsius. In addition, C. neoformans does not assimilate lactose and nitrates or produce pseudomycelia on cornmeal or rice-Tween agar. Most strains of C. neoformans can use creatinine as a nitrogen source, which may partially explain the growth of the organism in creatinine rich avian feces. Another useful biochemical characteristic of C. neoformans that distinguishes it from non-pathogenic strains is its ability to produce melanin. The fungal enzyme phenol oxidase acts on certain substrates like dihydroxyphenylalanine and caffeic acid to produce melanin.
Cryptococcus neoformans is an encapsulated yeast that was first described in an 1894 paper presented to the Greifswald Medical Society by Busse, a pathologist. Busse isolated the yeast from the tibia of a 31 year old woman, noted its resistance to sodium hydroxide, and published the case that same year. The following year Buschke, a surgeon, reported the same isolate from the same patient. Thus, the early eponym of Busse-Buschke’s disease. This one case served not only to identify a new yeast, but also to prove its pathogenic potential.
The exact pathogenesis of infection is still unclear. Pulmonary involvement is a common early event, and the organism is consequently assumed to gain access to the host via the respiratory route. In the absence of normal T-cell function, it then disseminates widely throughout the body, but especially to the central nervous system. Thus, most AIDS patients infected with C neoformans develop meningitis, although acute cryptococcal infection of almost every other organ has been described. Since the initial reports, the diverse spectrum of host responses to cryptococcal infection has become apparent. The spectrum ranges from harmless colonization of the airways and asymptomatic infection of laboratory workers resulting in a positive skin test to meningitis or disseminated disease. Although virulence for animals and possibly humans varies among strains of cryptococci, virulence probably plays a relatively small part in determining the outcome of an infection. The crucial factor appears to be the immune status of the host. The most serious infections usually occur in individuals with defective cell mediated immunity, such as the Acquired Immunodeficiency Syndrome (AIDS), organ transplantation, reticuloendothelial malignancy, corticosteroid treatment (but not neutropenia or immunoglobulin deficiency) and sarcoidosis. With the global emergence of AIDS, the incidence of cryptococcosis is increasing and now represents a major life threatening fungal infection in these patients. In the United States, 7% to 15% of patients with AIDS develop cryptococcal infectious. However, in some parts of sub-Saharan Africa coinfection with HIV and Cryptococcus neoformans approaches 90%.
Pathophysiology: Of the 19 species which comprise the genus Cryptococcus, only Cryptococcus neoformans is associated with human disease. Much of our understanding of the pathogenesis and the host defense mechanisms involved in C. neoformans infections comes from the studies of animal models. The organism is primarily transmitted by the respiratory route and not directly from one human to another. After being inhaled, C. neoformans is ingested by the alveolar macrophages. Unencapsulated yeast cells are readily phagocytosed and destroyed, whereas encapsulated organisms are more resistant to phagocytosis. Cryptococcal polysaccharide capsule has anti-phagocytic properties and may be immunosuppressive. The anti-phagocytic properties of the capsule block recognition of the yeast by phagocytes and inhibit leukocyte migration into the area of fungal replication.
The host’s response to Cryptococcal infection includes both cellular and humoral components. Animal models have demonstrated that natural killer cells participate inthe early killing of cryptococci and that antibody dependent cell mediated killing may be operative as well. In vitro, monocyte derived macrophages, natural killer cells, and T lymphocytes can inhibit or kill cryptococci. A successful host response includes an increase in helper T cell activity, skin test conversion, and a reduction in the number of viable organisms in the tissues. In addition to cellular mechanisms, anticryptococcal antibodies and soluble anticryptococcal factors have been described. Antibodies to cryptococcal antigen and complement play a critical role in enhancing the macrophage and lymphocyte mediated immune response to the organism. Monoclonal antibodies to capsular polysaccharide have been used to passively immunize mice against C. neoformans.
C. neoformans can cause an asymptomatic pulmonary infection followed later by the development of meningitis, often the first indication of the disease. If limited to the lungs, C. neoformans may cause pneumonia, poorly defined mass lesions, pulmonary nodules, and rarely pleural effusion. Although immune defects are common in patients with meningitis or disseminated infection, patients with disease confined to the lungs are usually immunocompetent.
Previously thought that the capsule promotes virulence by evading phagocytosis, based on the observation that phagocytic cells rarely ingest encapsulated cells without opsonins in vitro and on pathological studies that frequently show extracellular yeast cells in tissue. C. neoformans was suspected of being capable of intracellular pathogenesis in vivo, but according to the journal and any research I found, had never been demonstrated. This study attemted to answer the question of whether C. neoformans was a facultative intracellular pathogen by studying pulmonary infection in mice using light microscopy and electron microscopy. The fungus was studied over the course of the infection, and both capsular and acapsular stains were compared.
Methods and Materials:
Mice were infected intratrachealy with serotype D strain of C. neoformans variety neoformans which is capsulated, as well as with an acapsulated strain. The mice were then killed after various time intervals and the lung tissues examined using electron microscopy as well as light microscopy. This gave an accurate localization of yeast cells, and allowed visualization of cellular components.
The study showed that the capsule did not prevent phagocytosis in vivo, since cells survived and replicated inside macrophages, despite phagolysosomal fusion.
Initial course of pulmonary infection in mice involves rapid phagocytosis of C. neoformans cells, macrophage toxicity, neutrophil influx, and cellular disruption. C. neoformans intracellularly was associated with cytotxicity to host phagocytic cells. At 2 hours after infection, macrophages with intracellular yeast appeared intact, Many phagocytotic vacuoles with intracellular cells had discontinuous membranes by 4 hours, and by 8 hours after infection, the cell membrane of some macrophages with intracellular yeast was disrupted, a result consistent with cell destruction as a consequence of cytotoxic qualities of the yeast.
In areas of the lung in which large numbers of macrophages were present, there appeared to be two types of cells- macrophages with large numbers of yeast cells in which the macrophage appeared to have an increase of vacuoles in their cytoplasm. In these cells the extensive vacuolation resulted in cytoplasmic disruption of the macrophage.
The other type of cell present contained single yeast cells but where comparatively intact.
No funal cell components responsibele for macrophage cytotoxicity were characterized.
Volume and Phagolysosomal Fusion:
Phagolysosomal fusion was observed in alveolar macrophages 2 hours after infection. Acid phospphatase acitivity at the periphery of some phagosomes demonstrated phagosysosomal fusion.
The average phagosome volume increased with the time of infection, reflecting an increase in the average capsule size of C. neoformans cells in the lungs.
Yeast cells were found in phagosomes, lysosomes fuse with the pahgosome, and phagosomal acidification appeared to enhance the growth of C. neoformans. This provides a growth advantage to yeast cells in lung tissue.. The capsule separates the phagolysosomal membrane from the yeast cell wall, and this separation could limit the fungicidal effects of lysosomal products.
Intracellular replication : During the 1st 24 hours, most phagosomes contained 1 organism, after this period, the # of C. neoformans cells per phagosome and the # of phagosomes per macrophage increased (Refer to Figure).
The location of C. neoformans cells in the llung is a function of the time of infection. They are found primarily inside alveolar macrophages shortly after infection, but by 24 hours, the majority of cells were in the extracellular space. This is consistent with the intracellular replication followed by phagocytic cell lysis and release of live yeast into the extracellular space. The possibility of intracellular growth followed by cell lysis is supported by the present observation of cell debris in close proximity to yeast cells and also to reports that C. neoformans can replicate and lyse macrophage lineage cells in vitro. The transition from intracellular predominance to extracellular predominance was associeated with macrophage cytotoxicity and disruption, as indicated by 1)low cytoplasmic electron density and membrane disruption and 2)the appearance of cell debris in the alveolar space in close proximity to extracelllular yeast cells.
Capsular Vs. Acapsular:
With the acapsular strain, almost all cells were found intracellularly in macrophages 24 hours after infection. Macrophage cytoplasm and nuclei appeared normal. After 14 days, lungs of mice appeared normal and no organisms were seen, indicating a clearance of pulmonary infection.
In contrast, the capsular strain, the macrophages contained multile phagosomes with more than one yeast cell, and the macrophage cytoplasm contained vacuoles. There appeared to be intracellular polysaccharide synthesis, and the capsule may contribulte to intracellular survival by providing a buffer space between the site of lysosome-phagosome fusion and the fungal cell wall. At 24 hours after infection, the distance between the phagosome memebrane and the cell wall for phagocytosed encapsulated strains was 8.6 X that for the nonencapsulated strain.
In vitro studies were also done to confirm that encapsulated, but not acapsular, yeast cells replicated intracellularly and were toxic to phagocytic cells. With increased incubation times, the number of yeast cells per phagocytic cell increased for the encapsulated, but not the acapsular strain.
intracellular localization has been associated with both control and persistence of infection
The results demonstrated that intracellular residence was associated with multiple yeast cells per phagosome, a higher budding index, and the emergence of a yeast cellpopulation that was heterogeneous in size. This is indicative of intracellular replication in vivo.